Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness

Abstract

Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. All these interactions have the potential to alter the frequencies of mtDNA haplotypes (mitotypes) in nature and may impact human health. In a model laboratory system, we fed four diets varying in Protein: Carbohydrate (P:C) ratio (1:2, 1:4, 1:8 and 1:16 P:C) to four homoplasmic Drosophila melanogaster mitotypes (nuclear genome standardised) and assayed their frequency in population cages. When fed a high protein 1:2 P:C diet, the frequency of flies harbouring Alstonville mtDNA increased. In contrast, when fed the high carbohydrate 1:16 P:C food the incidence of flies harbouring Dahomey mtDNA increased. This result, driven by differences in larval development, was generalisable to the replacement of the laboratory diet with fruits having high and low P:C ratios, perturbation of the nuclear genome and changes to the microbiome. Structural modelling and cellular assays suggested a V161L mutation in the ND4 subunit of complex I of Dahomey mtDNA was mildly deleterious, reduced mitochondrial functions, increased oxidative stress and resulted in an increase in larval development time on the 1:2 P:C diet. The 1:16 P:C diet triggered a cascade of changes in both mitotypes. In Dahomey larvae, increased feeding fuelled increased β-oxidation and the partial bypass of the complex I mutation. Conversely, Alstonville larvae upregulated genes involved with oxidative phosphorylation, increased glycogen metabolism and they were more physically active. We hypothesise that the increased physical activity diverted energy from growth and cell division and thereby slowed development. These data further question the use of mtDNA as an assumed neutral marker in evolutionary and population genetic studies. Moreover, if humans respond similarly, we posit that individuals with specific mtDNA variations may differentially metabolise carbohydrates, which has implications for a variety of diseases including cardiovascular disease, obesity, and perhaps Parkinson's Disease.

Fig 1. Population cages, larval development and adult fecundity of four mitotypes.

Population cage studies, numbers of flies eclosing in 3 d, and egg counts show that larvae harbouring Alstonville mtDNA had an advantage on the 1:2 P:C diet while those with Dahomey mtDNA had the advantage when fed 1:16 P:C food. (A) Initial studies competed D. melanogaster w¹¹¹⁸ flies harbouring four mitotypes fed four Protein: Carbohydrate (P:C) diets (n = 3 cages/diet). The mitotypes were Alstonville, Dahomey, Japan and w¹¹¹⁸. The P:C ratios were 1:2 (top left), 1:4 (top right), 1:8 (bottom left) and 1:16 (bottom right). The mitotype of 4,608 flies was determined by amplifying ~900bp of mtDNA and then Sanger sequencing. After 12 generations, Alstonville had the highest frequency on the 1:2 P:C diet while Dahomey was highest on 1:16 P:C food. Flies with Japan or w¹¹¹⁸ mtDNA declined in frequency in all diets. Symbols show mean± s.e.m. (B) Eclosion percentage in a 3 d window for four mitotypes fed the 1:2 P:C or 1:16 P:C diet (n = 6 bottles/mitotype/diet) with each bottle seeded with 214.5± 14.2 eggs/bottle. t-tests compared Alstonville with Dahomey (see text). (C) Flies from each mitotype were assayed for fecundity on the 1:2 P:C and 1:16 P:C diets by egg count over 3 d, with an average of 32 flies/mitotype/diet. t-tests compared Alstonville + Dahomey with Japan + w¹¹¹⁸ (see text). Bars show mean± s.e.m. * p< 0.05, ** p< 0.01, *** p< 0.001.

Fig 2. Population cages and larval development of two mitotypes.

(A) Flies with Alstonville or Dahomey mtDNA were competed (n = 3 cages). Initially, the diet had a 1:2 P:C ratio. After four generations, the diet was switched to 1:16 P:C. An incubator malfunction killed all Drosophila in generation 16, so cages were re-established with a similar frequency of each mitotype. In generation 20, the diet was swapped back to 1:2 P:C. Plotted data are mean± s.e.m. mitotype/generation/cage/diet from ~80 flies/cage. (B) The number of females eclosing in 3 d was determined. The control was flies with w¹¹¹⁸ nuclear background fed 1:2 P:C and 1:16 P:C laboratory diets (n = 29 replicates of ~80 flies/rep for the 1:2 P:C diet, and n = 48 replicates of ~80 flies/rep for the 1:16 P:C diet). First, the nuclear genetic background was replaced with Oregon R (n = 6 biological rep/mitotype/diet). Second, the nuclear genetic background was replaced with Canton S (n = 5 biological rep/mitotype/diet). Third, passionfruit and banana replaced the laboratory diets (w¹¹¹⁸ nuclear background, P:C ratio of ~1:2 P:C and ~1:16 P:C, respectively; n = 5 rep/mitotype/diet). Finally, the microbiome obtained from wild-caught flies was introduced (w¹¹¹⁸ nuclear background, n = 6 biological rep/mitotype/diet). The 3 d eclosion window began on day 14 for 1:2 P:C diet and day 28 d for 1:16 P:C diet. Plotted data are mean± s.e.m. * p< 0.05, ** p< 0.01, *** p< 0.001 as calculated by t-tests (see text).

Fig 3. Quaternary and secondary structure modelling.

(A) The V161L site on TM6 was predicted to slow the cycling of the proton pump. The shorter arrow indicates the movement of TM5 as the Helix HL moves. (B) The mutation of site 161 from valine (left panel) to leucine (right panel) likely increases steric hindrance with tyrosine 184, narrowing the proton channel. (C) The secondary structure of the GTPase center in the lrRNA of D. melanogaster showing the site of mutation and structurally related residues.

Fig 4. The V161L ND4 amino acid change in complex I of Dahomey mtDNA influenced mitochondrial functions.

(A) The activity of complex I is higher in Alstonville larvae (n = 7 biological rep/mitotype/diet- with two failed reactions removed). (B) Oxygen consumption rate with complex I substrates is higher in Alstonville larvae (n = 6 biological rep/mitotype for 1:2 P:C diet; n = 9 biological rep/mitotype for 1:16 P:C diet). (C) Representative western blot showing reduced levels of complex I (CI) in Dahomey compared to Alstonville on both diets. There were no obvious differences in complex V (CV) or actin protein levels. Repeat gels showed all CI bands were the same size. (D) Native protein gel showing reduced activity of the peripheral arm of complex I (CI) and the supercomplex (SC) in larvae harbouring Dahomey mtDNA on both diets. Bars (mean± s.e.m). t-tests between mitotypes * p< 0.05, ** p< 0.01 (see text).

Fig 5. Corroboration that the complex I mutation in Dahomey drives the population cage results.

(A) Adding rotenone to the Alstonville diet created a Dahomey phenocopy. This phenocopy developed more quickly than controls when fed the 1:16 P:C food showing that partial inhibition of complex I was beneficial. Adding rotenone to the Dahomey fly food created a disease model and these larvae developed more slowly on both diets (n = 5 biological rep/mitotype/diet with and without rotenone treatment). (B) Complex I activity was decreased in the phenocopy, mimicking the Dahomey mitotype (n = 7 biological rep/mitotype/diet without rotenone treatment and 6 biological rep/mitotype/diet with rotenone treatment). (C) SOD activity increased in rotenone-treated larvae. On both foods, SOD activity in the phenocopy was not different from the Dahomey mitotype (n = 5 biological rep/mitotype/diet with and without rotenone treatment). (D) Larval development times of D. melanogaster harbouring the Madang (with the V161L ND4 mutation) and the Victoria Falls (without the ND4 mutation) mitotypes shows the same flip in development times as Dahomey and Alstonville (n = 6 bottles/mitotype/diet). Plotted data were mean± s.e.m. * p< 0.05 and ** p< 0.001, as calculated by t-tests (see text). Note: complete post-hoc analyses including all treatments for panels A-C are presented in S5 Fig.

Fig 6. Heat map showing relative expression of mtDNA encoded genes in third instar female wandering larvae harbouring Dahomey and Alstonville mtDNA that were fed on 1:2 P:C and 1:16 P:C ratio diets.

Within each treatment, there were four replicates (1–4). Log-expression values were batch corrected and standardised by gene. The darker the blue the more negative the Z score. The darker the red the more positive the Z score.

Fig 7. Transcriptomics and metabolomics assays.

Differentially expressed KEGG pathways and metabolites from wandering third instar female larvae fed the 1:2 P:C and 1:16 P:C food. Red indicates elevated in Dahomey, blue elevated in Alstonville with darker colours representing smaller FDRs. Detailed FDRs are shown in S3 Table. (A) The differentially expressed KEGG pathways observed in RNA-seq profiling for the 1:2 P:C diet (n = 4 biological rep/mitotype). MXC P450 is Methoxychlor-Cytochrome P450, C P450 is Cytochrome P450, A & A is Ascorbate and Aldarate, P & C is Porphyrin and Chlorophyll, P & G is Pentose and Gluconate, ER is endoplasmic reticulum. (B) The differentially expressed KEGG pathways observed in RNA-seq profiling for the 1:16 P:C diet (n = 4 biological rep/mitotype). NER is nucleotide excision repair, BER is base excision repair, FoxO is Forkhead box, HR is homologous recombination, Pyrimidine is pyrimidine metabolism, OXPHOS is oxidative phosphorylation, TCA is tricarboxylic acid, Glycerophospholipid is glycerophospholipid metabolism, and Carbon is carbon metabolism. Detailed FDRs are shown in S5 Table. (C) Differentially abundant metabolites observed in GC/MS profiling (n = 5 biological rep/mitotype) for the 1:2 P:C diet. (D) Differentially abundant metabolites observed in GC/MS profiling (n = 5 biological rep/mitotype) for the 1:16 P:C diet.

Fig 8. Basal ROS, antioxidant expression, mtDNA copy number and ATP levels Alstonville larvae had an advantage when fed the 1:2 P:C diet as the V161L ND4 amino acid change in complex I of Dahomey reduced the efficiency of ATP production.

(A) Measurement of basal ROS shows higher levels in Dahomey fed the 1:2 P:C diet. ROS levels were similar when larvae were fed the 1:16 P:C diet (n = 9 biological rep/mitotype on the 1:2 P:C diet, and 8 biological rep/mitotype on the 1:16 P:C diet with 2 failed reactions in Alstonville). (B). GstE1 and GstE5 expression was highest in Dahomey larvae fed the 1:2 P:C diet (n = 6 biological rep/mitotype/diet for both genes). (C) Alstonville larvae had higher mtDNA copy number when fed the 1:2 P:C diet but both mitotypes had equivalent and lower copy number when fed the 1:16 P:C diet. MtDNA copy number show the relative expression of ND4 (ND4/Actin) and lrRNA (lrRNA/Rp49) (n = 8 biological rep/mitotype/diet with 2 failed reactions for ND4/Actin and 7 biological reps/mitotype/diet for lrRNA/Rp49 with 2 failed reactions). (D) Total cellular ATP levels were higher in Alstonville larvae fed the 1:2 P:C diet but were similar when fed the 1:16 P:C diet suggesting a mitohormetic response (n = 8 biological rep/mitotype/diet, with two failed reactions) Bars show mean± s.e.m. t-tests between mitotypes * p< 0.05, ** p< 0.01 (see text).

Fig 9. Proposed metabolic differences between Drosophila larvae fed the 1:2 P:C food.

Development time for Alstonville larvae was faster than Dahomey because the V161L ND4 mutation in Dahomey caused reduced flow through the electron transport system. Red indicates elevated in Dahomey, blue higher in Alstonville. The mutation created a backup of glucose, which was likely metabolised to hexose, trehalose, and D-maltose. Lactate and alanine were also elevated. The mutation also caused an increase in ROS production, which resulted in an oxidative stress P450 response (including elevated levels of GstE1 and GstE5), high SOD activity and a decrease in ATP level.

Fig 10. Tests of hypotheses using other sugars and inhibitors.

Dietary modification of the 1:16 P:C diet with replacement of sugars (sucrose was the dietary sugar for the Control) and inhibitors. Replacement sugars were sorbitol, fructose, mannose, fucose, xylose, and gluconate (n = 4 rep/mitotype).The inhibitors were Epalrestat (Polyol pathway) (n = 5 rep/mitotype), and Etomoxir (β -oxidation) (n = 5 rep/mitotype). More Dahomey than Alstonville flies eclosed in a 3 d window when fed the control diet, as well as diets containing sorbitol, fructose, mannose, and fucose. Fewer Dahomey flies eclosed in a 3 d window when fed gluconate. There was no difference in the number of flies eclosing in 3 days between mitotypes when xylose was the dietary sugar or when Epalrestat or Etomoxir was added to the diet. * p< 0.05; ** p< 0.01, *** p<0.001 as determined by t-tests (see text). Dunnett’s tests compared Dahomey females fed the control diet to diets supplemented with inhibitors or the control diet compared with other sugars • p< 0.05 (see text).

Fig 11. The polyol pathway is upregulated in Dahomey larvae fed the 1:16 P:C diet.

(A) Expression of N and CrebB differed when larvae were fed the control (sucrose) diet, or sorbitol was the dietary sugar, but differences were lost when Epalrestat was added to the diet (n = 6 rep/mitotype, with n = 8 for Epalrestat fed Dahomey). (B) Food eaten was higher in Dahomey larvae than in Alstonville larvae. Food consumption increased when sorbitol was the dietary sugar and decreased when Epalrestat was added to the diet (n = 12 larvae/mitotype/diet were added to dye labelled food. Larvae with food visible in guts were collected and analysed: control-Alstonville = 7 larvae, control-Dahomey = 9 larvae, sorbitol-Alstonville = 8 larvae, sorbitol-Dahomey = 11 larvae, Epalrestat-both mitotypes = 10 larvae. Bars show mean ± s.e.m. * p< 0.05 and ** p< 0.01, as calculated by t-tests (see text).

Fig 12. β-oxidation of fatty acids is upregulated in Dahomey larvae fed the 1:16 P:C diet.

(A) Triglyceride levels were higher in Dahomey larvae fed the control diet. When Etomoxir (Eto) was added to the control diet, triglyceride levels increased, and differences between the mitotypes was lost (n = 14 rep/mitotype/treatment with 6 failed reactions). (B) Expression of eloF) and bmm) were higher in Dahomey larvae fed the control diet. Differences were lost when Etomoxir (Eto) was added to the control diet (n = 6 rep/mitotype). (C) β-oxidation activity was highest in Dahomey larvae (n = 10 biological rep/mitotype–with one outlier removed). (D) Acetyl-coA enzyme activity in the cytosol and extracted mitochondria was higher in Dahomey larvae. (n = 9 biological rep/mitotype–with four outliers removed from the cytosol data). (E). NAD⁺/NADH ratio was higher in Dahomey larvae (n = 7 rep/mitotype). (F) Starvation survival was greatest in Dahomey larvae (n = 56 for Alstonville and 91 for Dahomey). Bars (mean ± s.e.m). p< 0.05 and p< 0.01, as calculated by t-tests (see text).

Fig 13. Proposed mitohormetic responses in Drosophila larvae fed the 1:16 P:C food (red indicates elevated in Dahomey, blue higher in Alstonville).

The mitohormetic response, involving at least two separate pathways, enabled Dahomey to develop faster than Alstonville larvae. First, larvae with Dahomey mtDNA ate more, which caused third instar larvae to weigh more. Backup of sugars produced increased activity of the polyol pathway and increased N expression. Increased N expression blocked CrebB and fed back to increase food consumption. Second, pyruvate was metabolised to acetyl-CoA and exported from the mitochondrion for fatty acid synthesis and palmitic acid and stearic acid levels increased. The long-chain fatty acids were catabolised by β oxidation, resulting in the formation of NADH and FADH₂. FADH₂ shuttled electrons to the quinone pool and partially by-passed ETC complex I where the V161L mutation occurred. In contrast, Alstonville larvae upregulated glycogen metabolism and activity of the pentose phosphate pathway increased. Increased glycogen metabolism increased wandering, which diverted energy away from development. Increased insulin signalling decreased larval food consumption.

Fig 14. Glycogen metabolism is increased and the pentose phosphate pathway is upregulated in Alstonville larvae fed the 1:16 P:C diet.

(A) Glycogen level was highest in Alstonville larvae (n = 10 biological rep/mitotype). (B) Physical activity was highest in Alstonville larvae fed the control (sucrose) diet and when gluconate was the dietary sugar (n = 16 larvae/mitotype with 3 outliers removed when fed sucrose and 12 larvae/mitotype when fed gluconate). (C) Expression of Zw and the Ilp2 on control (sucrose) and gluconate diets were higher in Alstonville larvae (n = 6 rep/mitotype with 1 failed reaction). Bars (mean ± s.e.m). * p< 0.05 and ** p< 0.01, as calculated by t-tests (see text).